Glycolytic Pathway of Trypanosoma Brucei Brucei Hexokinase 1 Gene in Trypanosoma Brucei Brucei TREU 927 and T. Brucei Gambiense

Journal of Medical and Applied Biosciences Volume 10, ISSN: 2277 - 0054 Number 1, 2018

MOLECULAR DETECTION OF HEXOKINASE 1 GENE IN THE BLOODSTREAM FORM OF TRYPANOSOMA BRUCEI BRUCEI (FEDERE ISOLATE)

Ishaya Y. Longdet1* and Rotimi J. Ojo1,2 1Department of Biochemistry, University of Jos, Plateau State, Nigeria 2Department of Biochemistry, Bingham University, Karu, Nasarawa email: [email protected]; [email protected]

ABSTRACT Hexokinase 1 (EC:2.7.1.1) which belongs to the Hexokinase 2 superfamily is the first key regulatory enzyme in the glycolytic pathway of Trypanosoma brucei brucei. Therefore the enzyme has great significance in metabolism of the parasite particularly for the purpose of generating ATP and nucleic acid precursors. There is increasing interest in the study of enzymes in parasites for various purposes. This research therefore attempted to detect the presence of the hexokinase 1 gene in Trypanosoma brucei brucei (Federe isolate) which is prevalent in Nigeria. The parasites were grown in rats, harvested and purified using diethyl amino ethyl (DEAE) cellulose chromatography. From the genomic DNA isolated the hexokinase 1 gene was amplified through Polymerase Chain Reaction. The amplicon was sequenced and studied using some Bioinformatics tools. The 1401bp gene sequence (Accession number MH 198230) obtained translated into 464 amino acid sequence. The primary nucleotide sequence of the gene compares well with Trypanosoma brucei TREU 927 and T. brucei gambienseDAL972. The predicted molecular mass of the polypeptide as calculated using ExPASy is 51,918.50Da (~52kDa) giving the hexameric protein a molecular weight of around 311.5kDa which differs from the mammalian hexokinase The Isoelectric point (pI) was

Journal of Medical and Applied Biosciences Volume 10, Number 1, 2018 predicted as 9.64 indicating that at neutral pH, the enzyme has a net positive charge. These revealed that the T.b.brucei hexokinase 1 is active in the bloodstream form of the parasite. Keywords: Hexokinase 1, Trypanosoma brucei brucei, gene sequence

INTRODUCTION pathway which serves as the African trypanosomiasis is only source of ATP and mainly caused by nucleic acid precursors for Trypanosoma brucei species the parasite in the infective and is estimated to affect stage and differs from the about 70 million people in host enzyme. The parasites Africa. This disease results in the mammalian in great loses. It has been bloodstream depend solely estimated that about $ 4.5 on glycolysis because of its billion is loss annually from poorly developed animal fatality (Franco et mitochondrion which lacks al., 2014; Tesfaye et al., active components of the 2012; Simarro et al., 2012). citric acid cycle and the It has also been reported electron transport chain that the disease brings (Joice et al., 2013). This about death if not treated study was designed to (Franco et al., 2014). detect the hexokinase 1 Therefore there is a need gene of T.b.brucei (Federe to further understand the isolate) as a potential biochemistry of the target for drug or vaccine parasite. One of the development. biochemical elements in T. b.brucei, hexokinase 1, is the first key regulatory enzyme of the glycolytic

Molecular Detection of Hexokinase 1 Gene in the Bloodstream Form of Trypanosoma Brucei brucei (Federe Isolate)

MATERIALS AND Inc. Indonesia. Sequencing METHODS analyses were performed by Reagents Macrogen in Netherlands. The reagents used were of analytical grade and Growth of Trypanosomes in purchased from Sigma, BDH Rats POOLE, England, Whatman, The Trypanosoma b. brucei USA; Pharmacia Fine (Federe isolate) was Chemicals and Amresco Life obtained from the Science, Fountain Veterinary and Livestock Parkway,Solon. DNA Studies Department of the extraction kit was Nigerian Institute for purchased from ZYMO Trypanosomiasis Research, RESEARCH. Taq polymerase Vom, Plataeu State, Nigeria. and High Fidelity Healthy rats were lightly Polymerase Enzyme Mix anaesthetized and infected were bought from Promega, intraperitoneally with the USA and Fermentas, parasite (about 1x104 respectively. Molecular size parasites/ml) sourced from marker was purchased from tail vein blood of the Roche, Mannheim Germany. Trypanosoma brucei brucei Oligonucleotide primers infected donor rat were synthesized by Inqaba reservoir at a massive biotec Industry, Pretoria parasitemia (1x106 South Africa. The GeneAmp parasite/ml) in 0.3ml of PSG PCR System9700 for gene (pH 7.8). Parasitemia was amplification was obtained monitored daily from the from Applied Biosystems, second day after the Indonesia and the Gel infection by bleeding the Documentation System was tail of each animal and the obtained from Synegene® blood observed under X400

Journal of Medical and Applied Biosciences Volume 10, Number 1, 2018 magnification of a phase aminoethyl cellulose (DEAE) contrast microscope with anion exchanger column fields examined and the (DE-52, Whatman) as mobile parasites counted. described by Lanham and Godfrey (1970). The eluate Purification of Bloodstream containing the trypanosomes Form T. brucei brucei in centrifuge tubes were from Rat centrifuged for 10mins at At peak parasitemia, the 1600 x g in a bench top circulating blood was centrifuge, and the withdrawn by cardiac supernatant was discarded. puncture under terminal The trypanosome pellets anaesthesia using a syringe were then washed by gentle loaded with a small volume resuspension in 1ml of PBS of PSG containing heparin. (pH 7.4) and transferred to The whole blood was microcentrifuge tubes and centrifuge at 1,250 × g for centrifuged for 5mins at 15 minutes at 4oC resulting 1600 x g. After a final in separation of the sample 5mins centrifugation at into three different layers. 1600 x g, the supernatant The white Buffy coat was removed, the containing the parasite trypanosomes were pooled formed between the red and the pellet was blood cells and the plasma resuspended in PBS (pH 7.4) layer. The Buffy layer was for subsequent analysis. carefully removed using a Pasteur pipette and re- DNA Isolation and suspended in a small volume Polymerase Chain Reaction of phosphate buffered (PCR) saline with glucose (PSG, pH The genomic DNA was = 7.8) and thereafter extracted from the purified using diethyl resulting trypanosome cell

Molecular Detection of Hexokinase 1 Gene in the Bloodstream Form of Trypanosoma Brucei brucei (Federe Isolate) pellets using the ZR of amplification in a Fungal/Bacterial DNA thermocycler using the MiniPrep (ZYMO following Amplification RESEARCH) following conditions: Polymerase manufacturer’s instruction. activation 94°C for 5 min; The following primers Denaturation 94°C for 30 s; (Forward 5’- Annealing 48°C for 30 s; ATGTCTAGACGCCTAAACA Extension 72°C for 1 min. ATATCC-3’ and Reverse 5’- Step 2-4 were repeated for TTACTTGTCGTTCACCACCA 30 cycles and Final TTGCG-3’) which were extension was carried out at designed based on the 72°C for 5 minutes. The PCR sequence obtained from product was then subjected GeneDB were used to to 1.5% agarose gel amplify the gene sequence electrophoresis (1.5 % w/v) of T. brucei brucei and the results documented hexokinase 1 gene. The PCR using Gel Documentation reaction was performed in a System (Synegene® Inc. 50μl reaction mixture which Indonesia). contained 27μl of nuclease free water, 10 μl of 5 X DNA Sequencing and phusion buffers, 1 μl of Bioinformatics Analysis dNTPS, 1.5μl of DMSO, 2.5 The T. brucei brucei μl of forward primer, 2.5 μl Hexokinase 1 DNA amplified of reverse primer, 0.5 μl of was then sent to macrogen Taq DNA polymerase in Netherlands for (Promega) and 5.0 μl of the sequencing. Sequence of genomic DNA of T.brucei isolated T. brucei brucei brucei. The tubes Hexokinase I was analyzed containing the mixture will using some BLAST tools be subjected to 30 cycles provided by NCBI

Journal of Medical and Applied Biosciences Volume 10, Number 1, 2018

(http://www.ncbi.nlm.nih.g (OFR) that is less than ov/).The physico-chemical 1650bp but far above parameters of T. brucei 1000bp (Figure 1), this was brucei Hexokinase I was thereafter confirmed by analyzed using ExPASy sequencing the gene which (http://www.expasy.org/). showed that it contained 1401bp (Figure 2). This size RESULTS AND was more than 1350bp DISCUSSION obtained by Chen et The result from the gel al.,(2014) for hexokinase electrophoresis showed from Clonorchis sinensis that the T. brucei brucei but closer to that of T. (Federe isolate) hexokinase cruzi (1416 bp) as reported 1 gene band (lanes S1 & by Cáceres et al.,(2003) . S2) has open reading frame

M 1 2

2000bp 1401bp 1000bp

Figure 1: Analysis of PCR amplicon on 1% agarose gel elctrophoresis of T. b.brucei Hexokinase 1 gene. S1 and S2= T. b. brucei Hexokinase 1 gene run in duplicate; M= Molecular marker

Molecular Detection of Hexokinase 1 Gene in the Bloodstream Form of Trypanosoma Brucei brucei (Federe Isolate)

The NCBI BAST of the sequence yielded a good similarity (88%) with hexokinase sequences of genes of Trypanosoma brucei brucei TREU927(AJ437577.1); Trypanosoma brucei Gambiense(FN554973.1) ; Trypanosoma cruzi (XM841212.1). The Taxonomy report shows that the gene sequence is of an organism that is a Trypanozoon summarised in Table 1.

ATGTCTAGAC GCCTAAACAA TATCCTCGAA CACATCTCGA TCCAGGGAAA TGATGGTGAG ACTGTGCGTT CTTTTTATGT TACGTTGCAC GCCCTCACCA ACCAACCTTT TCTGAGTGTC GAGTCTATGC GACAGATCAT GACATACCTC CTGTACGAGA TGGTGGAGGG TCTTGAGGGT CTTCACACCA CCCTCTCCAT TTTACCATGT ACCCGACGCG GACCCTACGC CCTGGCCTTC TTCTCCCCCT TTGACCTCTG TGGTTCCATT TTCTGTGTGT TGCGTGGCCA TGCAAGGAGG GTCCCGTGGT GTTTTCCTCT CCTTCTGCAT TCAAAATCCC CAAATATGCC CTTGAGGGTA ACGCCACCGA TCTGTTTGGC TTCATTGCAT CCAATGTGAA AAAACCCCTG GAACTCTCGT ACCATGGAAA CTCGTGCACC TGAGGACCTC AATCGCACAG TTCCTCTTGG GTTTACCTTC AGTTTCCCCG TGGAGCAGAC GAAGGTTAAC CGTGGTGTGC TTATCCGGTG GACGAAGGGC TTCAGCGGAT GCTATGTGAT TGCCCTTCTT CGCGCTGCTT TTGGGAAATT TAGCCTAAGT GTCAATGTTG TGGCTTTGTG CAACGACCCC TTTTGAACTT TAATTTCGCA TTACTTTAAG GACCCTGAGG TACAGGTTGG TGTGATTATC GGCACTGGTC CAGATGCGTG CTACTTTGAC AGGGCCTCTG CTGTGACAAA AGACCGTGCC TTTGCTGCTC GTGAGTCAGC ACTTACTCCC ATCAATATAC AAAGCCTCCA TTTTGACTCC GAGTACCGGT ATGTCCTCTA CAACAAATTT CAACTTGTAT ATCGACGATC CGTCGTTAAA CAAATGTCAA CATGCTCTAG AGAAGATATA TCCCGCATGT ATCGCGGTAA AATCTCCCGC CGCGTTATTG TGCACCTTTC GTCTATAAGC CGCCTTCCTG CGGCACTCCA GACGGCTTTT GGACAACCGG GTGTCGTTTT AATCCCCATT TCCCCGGAAT CACCAGTGCT TCACCTTAAG CCCCGACTTC AGTTCACTCG CAGCACGATC CAGAAGGTGT GTGGTGTTGA CGTGCAGTCA ATTGAAGACC TTCGCATCAT TCGCGATGTG TGCCGCCTTG TCCGTGGGAG GGCTGCGCAA CTCTCTGCTT CCTTCTGCTG CGCTCCACTG GTTAAGACTC AAACACAGGG CCGTGCAACT ATTGCAATTG ACGGCTCCGT GTTTGAGAAG ATTCCGTCAT TCCGCCGCGT CTTGCAGGAC AACATCAACC GTATCCTTGG CCCTGAGTGC GATGTCAGGG CCGTTCTCGC AAAGGATGGC AGTGGAATTG GTGCTGCATT TATTTCCGCA ATGGTGGTGA ACGACAAGTA A

Figure 2: Primary Nucleotide Sequence of Trypanosoma brucei brucei (Federe isolate) hexokinase 1 gene (Accession number MH 198230).

Journal of Medical and Applied Biosciences Volume 10, Number 1, 2018

Table 1: BLAST Results Showing Hexokinase 1 Sequence with significant alignments Description of Organism Max Total Query E identity Accession score score cover value Number Trypanosoma brucei gambiense 0.0 DAL972 hexokinase, putative partial 1626 1626 100% 88% XM_011779121.1 mRNA Trypanosoma brucei gambiense 0.0 DAL972 chromosome 10, complete 1626 3145 100% 88% FN554973.1 sequence Trypanosoma brucei brucei strain 0.0 927/4 GUTat10.1 hexokinase 1620 1620 100% 88% XM_817363.1 (Tb10.70.5820) partial mRNA Trypanosoma brucei HK gene for 0.0 1620 1620 100% 88% AJ345044.1 hexokinase Trypanosoma brucei gambiense 0.0 DAL972 hexokinase, putative partial 1519 1519 98% 87% XM_011779123.1 mRNA Trypanosoma brucei brucei strain 0.0 927/4 GUTat10.1 hexokinase 1507 1507 98% 87% XM_817364.1 (Tb10.70.5800) partial mRNA

The translated amino acids sequence (Figure 3) gives 464 amino acids, which compares well with 471 amino acids of T. brucei brucei hexokinase 1(XP_822456.1 ) in the NCBI data base and 474 amino acids sequence obtained by Willson et al.,(2002).

MSRRLNNILE HISIQGNDGE TVRSFYVTLH ALTNQPFLSV ESMRQIMTYL 50 LYEMVEGLEG LHTTLSILPC TRRGPYALAF FSPFDLCGSI FCVLRGHARR 100 VPWCFPLLLH SKSPNMPLRV TPPICLASLH PMKNPWNSRT METRAPEDLN 150 RTVPLGFTFS FPVEQTKVNR GVLIRWTKGF SGCYVIALLR AAFGKFSLSV 200 NVVALCNDPF TLISHYFKDP EVQVGVIIGT GPDACYFDRA SAVTKDRAFA 250 ARESALTPIN IQSLHFDSEY RYVLYNKFQL VYRRSVVKQM STCSREDISR 300 MYRGKISRRV IVHLSSISRL PAALQTAFGQ PGVVLIPISP ESPVLHLKPR 350 LQFTRSTIQK VCGVDVQSIE DLRIIRDVCR LVRGRAAQLS ASFCCAPLVK 400 TQTQGRATIA IDGSVFEKIP SFRRVLQDNI NRILGPECDV RAVLAKDGSG 450 IGAAFISAMV VNDK Figure 3: Translated amino acids sequence of T. brucei brucei (Federe isolate) Hexokinase 1. KEY: G - Glycine P – Proline, A – Alanine, V – Valine, L – Leucine, I – Isoleucine, M

Molecular Detection of Hexokinase 1 Gene in the Bloodstream Form of Trypanosoma Brucei brucei (Federe Isolate)

– Methionine, C–Cysteine, F – Phenylalanine,Y – Tyrosine, W – Tryptophan,H – Histidine, K - Lysine R – Arginine, Q – Glutamine, N – Asparagine, E - Glutamic Acid, D - Aspartic Acid S – Serine, T – Threonine

The molecular mass and pI (Ureta, 1982; Thompson et of the polypeptide were al., 1994; Willson et al calculated using ExPASy 2002). It was T. brucei software by Swiss Institute brucei hexokinase 1 gene of Bioinformatics that was detected and (Gasteiger et al., 2005). studied. The predicted molecular mass of the polypeptide ETHICAL APPROVAL which exist in hexameric Animal experiments were form in Trypanosomal carried out in accordance hexokinase is 51,918.50Da with the instructions for (~52kDa) giving it a the care and use provided molecular weight of around by the University of Jos, 311.5kDa which appear to be Nigeria . different from mammalian hexokinase which exist as REFERENCE monomer (types A, B and C, Besteiro, S., Biran, M., 96-111 kDa; type D Biteau, N., Coustou, V., (glucokinase), 47 - 55 kDa). Baltz, T., Canioni, P. & Hexokinase from T. brucei Bringaud F. (2002). can therefore be classified Succinate secreted by as non-vertebrate Trypanosoma brucei is hexokinases since produced by a novel and vertebrate hexokinases all unique glycosomal have a molecular mass of enzyme, NADH- approximately 100 kDa fumarate reductase. J

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Molecular Detection of Hexokinase 1 Gene in the Bloodstream Form of Trypanosoma Brucei brucei (Federe Isolate)

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Reference to this paper should be made as follows: Ishaya Y. Longdet and Rotimi J. Ojo (2018), Molecular Detection of Hexokinase 1 Gene in the Bloodstream Form of Trypanosoma Brucei brucei (Federe Isolate) J. of Medical and Applied Biosciences, Vol. 10, No. 1, Pp. 58-71